The biochemistry of carbon radicals remains poorly understood despite the unique value of carbon radicals as mechanistic probes and their immediate relevance to oxygen radical pathology. The work proposed here builds on the following key advances made during the expiring period of support: (a) validation of a rationale for the fact that radicals are almost invariably produced by peroxidases but rarely by monooxygenases, (b) successful expression of catalytically active horseradish peroxidase (HRP) and lactoperoxidase in a baculovirus system, and (c) initial exploration of the structure-activity relationships for horseradish peroxidase by site specific mutagenesis. The first goal of the proposed work is to further elucidate the function of HRP, the prototypical peroxidase, with emphasis on (a) the structural features that govern catalytic turnover and substrate specificity, (b) the mechanism of substrate free radical generation, and (c) the catalysis of monooxygenase reactions. The second goal is to extend our growing understanding of plant and fungal peroxidases to lactoperoxidase and myeloperoxidase, two physiologically important mammalian enzymes, with emphasis on (a) the nature and mechanism of covalent binding of the prosthetic heme group to the protein, (b) the location of the two oxidation equivalents in the 2-electron oxidized intermediate known as compound I, and (c) the features that make possible the oxidation of halide ions. The third goal is to explore aspects of the biological fates of peroxidatively generated radicals, particularly their reactions with proteins. The collective intent of these studies is to advance our understanding of the enzymes that form radical species and of the reactions of these radicals with proteins. The results should help to clarify tic role of peroxidatively generated radicals in toxicological processes and to uncover mechanisms for the suppression of such processes.